Summary

The whisker follicle has CD34-positive stem cells that migrate from their niche near the bulge along the glassy membrane to the whisker bulb, where they participate in the formation of the whisker shaft. Using immunohistochemistry, we found the glycoprotein tenascin-C in the fibrous capsule of mouse whisker follicles, along the glassy membrane and in the trabecular region surrounding keratin-15-negative, CD34-positive stem cells. The related glycoprotein tenascin-W is found in the CD34-positive stem cell niche, in nearby trabeculae and along the glassy membrane. Tenascin-W is also found in the neural stem cell niche of nearby hair follicles. The formation of stress fibers and focal adhesion complexes in CD34-positive whisker-derived stem cells cultured on fibronectin was inhibited by both tenascin-C and tenascin-W, which is consistent with a role for these glycoproteins in promoting the migration of these cells from the niche to the whisker bulb. Tenascin-C, but not tenascin-W, increased the proliferation of whisker follicle stem cells in vitro. Thus, the CD34-positive whisker follicle stem cell niche contains both tenascin-C and tenascin-W, and these glycoproteins might play a role in directing the migration and proliferation of these stem cells.

Introduction

Whisker follicles are complex tactile organs found in all mammals except humans (Muchlinski, 2010). Unlike hair follicles, whisker follicles are surrounded by two venous sinuses and a tough fibrous capsule. The whisker root sheathes form a bulge under the more superficial ring sinus, which is a source of keratin-15-positive keratinocyte precursor stem cells (Amoh et al., 2010). A second population of stem cells found near the bulge is positive for the neural stem cell marker nestin and CD34. It has been proposed that the CD34-positive stem cells migrate along the glassy membrane of the outer root sheath to the dermal papilla, where they contribute to the development of the whisker shaft (Amoh et al., 2010). These cells can be isolated and studied in culture by explanting the whisker bulge and associated trabeculae (Sieber-Blum et al., 2004; Sieber-Blum and Hu, 2008), and have the potential to differentiate into neurons and non-neuronal cells in vitro (Sieber-Blum et al., 2004; Wong et al., 2006; Amoh et al., 2008).

Tenascin-C is an extracellular matrix glycoprotein associated with epithelial–mesenchymal interactions and cell motility in the embryo, and in the adult with solid tumor stroma (Chiquet-Ehrismann and Tucker, 2012). Tenascin-C also surrounds migrating neural crest cells (Tucker and McKay, 1991) and morpholino-mediated knockdown of tenascin-C expression by the neural crest inhibits their migration (Tucker, 2001). Tenascin-W is the most recently characterized member of the tenascin gene family. Similar to tenascin-C, it is found in the stroma of many solid tumors (Brellier et al., 2012a). Tenascin-W is also expressed in developing and adult trabecular bone, and it promotes the migration and proliferation of osteoblasts in vitro (Meloty-Kapella et al., 2008).

Here, we report that both tenascin-C and tenascin-W are part of the niche surrounding CD34-positive stem cells in mouse whisker follicles, and we characterize the effects of tenascin-C and tenascin-W on these cells in vitro.

Results and Discussion

Tenascin-C and tenascin-W are expressed in the adult mouse whisker follicle

Tenascin-C was expressed widely in the adult mouse whisker follicle (Fig. 1A). By contrast, the distribution of tenascin-W was more restricted (Fig. 1B). Tenascin-W immunostaining was found in the glassy membrane near the bulge and in the extracellular matrix of the trabeculae between the bulge and the capsule. Tenascin-W was also present in the extracellular matrix of the outer conical body near the sebaceous gland and in the fibrous capsule surrounding the whisker bulb. Double-label immunohistochemistry staining for tenascin-C and tenascin-W showed extensive overlap in the fibrillar matrix of the trabecular region (Fig. 1C,D). This was in contrast to fibronectin in this region, which was found in a fibrillar network that was mostly distinct from the tenascin-C-positive fibrils (Fig. 1E). However, both tenascin-C and fibronectin immunostaining was found in the glassy membrane underlying the trabecular region.

Tenascin-C and tenascin-W are expressed in adult mouse whisker follicles. (A) Tenascin-C localizes to the capsule (C), glassy membrane (GM) and trabecular region (TR) near the bulge. (B) Tenascin-W is restricted to the capsule near the bulb, the glassy membrane, the trabecular region and the mesenchyme surrounding the sebaceous gland (SG). (C) The merged image. The box indicates the region shown at higher magnification in D and E. (D) Higher magnification of the trabecular region shows regions of overlap of tenascin-C and tenascin-W immunostaining (yellow, arrowhead) as well as fibrils that are positive for tenascin-W (arrow). (E) Fibronectin is found in the glassy membrane (arrowhead) and in the extracellular matrix of the trabecular region. (F) Tenascin-W is found in a narrow ring (arrows) surrounding facial hair follicles. (G) The bulge is stained with anti-keratin-15 (K15). The region where tenascin-W is found is indicated with an asterisk. CS, cavernous sinus; DP, dermal papilla; ORS, outer root sheath; RS, ring sinus; Rw, Ringwulst; S, whisker shaft. Scale bars: 200 µm (A–C); 25 µm (D,E); 50 µm (F,G).

Tenascin-C and tenascin-W also had distinctive patterns of expression in facial hair follicles (Fig. 1F,G). Tenascin-C was found in the matrix surrounding the keratin-15-positive bulge (Kloepper et al., 2008), whereas tenascin-W immunoreactivity was limited to a narrow ring between the bulge and the sebaceous gland. This ring corresponds to the region where nestin-positive neural stem cells have been localized (Amoh et al., 2010).

Tenascin-C and tenascin-W are found in the extracellular matrix surrounding CD34-positive, keratin-15-negative stem cells

To determine the relationships between tenascin-C and tenascin-W and different populations of stem cells of the whisker follicle, sections were double labeled with antibodies against either tenascin-C and keratin-15 (Fig. 2A–C) or tenascin-W and CD34 (Fig. 2D–F). The anti-keratin-15 antibody labeled the epidermal stem cell region of the bulge near the tenascin-C-positive matrix of the trabeculae. By contrast, CD34-positive cells were found in a cluster near the junction of the trabecular region and the bulge that closely corresponded with the anti-tenascin-W-stained extracellular matrix. Because tenascin-C and tenascin-W largely colocalized in this region (Fig. 1D), both tenascin-C and tenascin-W are part of the CD34-positive stem cell niche. Both tenascin-C and tenascin-W were found in the fibrous capsule surrounding the bulb (Fig. 1C; Fig. 2G,H). Tenascin-C was also found in the glassy membrane near the bulb and tenascin-W was present near the CD34-positive cells that accumulate near the stalk of the dermal papilla (Fig. 2H). Anti-Ki67, a marker of cell proliferation, was used to demonstrate that this bulb was in anagen (Fig. 2I). The pattern of tenascin-C and tenascin-W immunoreactivity suggests that they might be able to influence the migration and proliferation of CD34-positive stem cells both in their niche and along their pathway to the whisker bulb. Tenascin-C and tenascin-W immunostaining was not observed in other epidermal stem cell niches (e.g. keratin-8- and keratin-17-positive dome cell niches in foot pads or dermis).

Colocalization of tenascin proteins and stem cells markers. (A) The keratin-15 (K15)-positive cells of the epidermal stem cell niche of the bulge. (B) Tenascin-C in the adjacent trabecular regions (TR). (C) Double-label immunohistochemistry showing the distinct, non-overlapping patterns of keratin-15 and tenascin-C. (D) CD34-positive stem cells in the trabecular region immediately adjacent to the bulge. (E) Tenascin-W in the trabecular region. (F) CD34-positive stem cells are embedded in a tenascin-W-rich extracellular matrix. (G) In the bulb, tenascin-C immunoreactivity is limited to the capsule (C) and the opening to the dermal papilla (arrows). (H) Tenascin-W immunoreactivity in the stalk of the dermal papilla (arrows), where CD34-positive stem cells are found (asterisk). (I) Anti-Ki-67 demonstrates that the bulb is in the actively growing stage of anagen. ORS, outer root sheath. Scale bars: 50 µm (A–F); 100 µm (G–I).

CD34-positive stem cells spread on fibronectin but fail to form stress fibers on tenascin-C and tenascin-W in vitro

Tenascin-C (Chiquet-Ehrismann and Tucker, 2012) and tenascin-W (Brellier et al., 2012b) interfere with cell spreading on fibronectin. The resulting lack of stress fibers and focal adhesion complexes is accompanied by increased cell motility. To test whether tenascin-C and tenascin-W have the potential to interfere with CD34-positive stem cell adhesion to fibronectin and therefore potentially promote their migration, whisker follicle bulges were explanted onto dishes or coverslips that were coated with fibronectin, fibronectin and tenascin-C, or fibronectin and tenascin-W. After 4–5 days, cells migrated from the bulge region onto the coated substrata (Fig. 3A). CD34-positive cells migrating on fibronectin had broad lamellae and were filled with TRITC-phalloidin-stained stress fibers that terminate in anti-vinculin-labeled focal adhesion complexes (Fig. 3B). By contrast, the CD34-positive cells that spread on mixtures of fibronectin and either tenascin-C (Fig. 3C) or tenascin-W (Fig. 3D) had small lamellae and relatively few stress fibers. There were significantly fewer focal adhesion complexes in stem cells cultured on a mixture of tenascin-C and fibronectin (P<0.05) or a mixture of tenascin-W and fibronectin (P<0.01) than in cells cultured on fibronectin alone (Fig. 3E). CD34-positive cells were also less spread (P<0.01) on mixtures of fibronectin and either tenascin-C or tenascin-W (Fig. 3F). Therefore, both tenascin proteins have the potential to promote the migration of stem cells along the glassy membrane to the whisker bulb. The distribution of tenascin-W in a gradient deep to the cavernous sinus, but not in the more superficial ring sinus, suggests that it could help guide the stem cells proximally to the whisker bulb.

Tenascin-C, but not tenascin-W, stimulates the proliferation of CD34-positive stem cells in vitro

To determine whether either of the two tenascin proteins has the potential to influence the proliferation of CD34-positive stem cells, bulges were explanted as described above. After 5 days, the cultures were fixed and immunostained with anti-Ki-67 antibody, which stains the nuclei of cells during the active phases of the cell cycle, but not the nuclei of cells during G0. Under the conditions described here, tenascin-C, but not tenascin-W, was able to increase the proliferation of CD34-positive stem cells in vitro (Fig. 3G). This is consistent with the results of others studying tenascin-C in stem cell niches. For example, Nakamura-Ishizu and co-workers (Nakamura-Ishizu et al., 2012) found that tenascin-C, which is part of the hematopoietic stem cell niche of bone marrow, promotes the proliferation of these cells in culture, and Yagi and colleagues (Yagi et al., 2010) found that tenascin-C increases the proliferation of neural stem cells in vitro. Tenascin-C might be a useful tool for those expanding populations of hair follicle-derived stem cells prior to their differentiation or use in therapeutic grafts.

Tenascin-C and tenascin-W are associated with CD34-positive stem cells in whisker follicles, and the effects of tenascin-C and tenascin-W on cultured CD34-positive stem cells are consistent with roles in promoting stem cell motility. Tenascin proteins, therefore, have the potential to play roles associated with the migration and proliferation of a population of cranial neural-crest-derived stem cells that have been proposed by others (Amoh et al., 2008; Amoh et al., 2010) to have significant potential therapeutic value.

Materials and Methods

Histology and immunohistochemistry

Frozen sections in the long axis of the mystacial macrovibrissal follicles from adult C57BL6 or 129/Sv mice were cut at 12–14 µm and air-dried. For immunohistochemistry, the sections were treated as described previously (Brellier et al., 2012b). Images were acquired with a Nikon Eclipse E800 or a Zeiss Axioscop fluorescence microscope.

Whisker follicle stem cells were cultured using an adaptation of published methods (Sieber-Blum and Hu, 2008). In brief, the dissected whisker fragment containing the stem cells was placed onto a plastic dish or coverslip coated previously with extracellular matrix (see below) with a small amount of medium and allowed to adhere for 1–2 hours. The dish was then gently filled with αMEM supplemented with 10% fetal calf serum and returned to the incubator. After 5 days, the whisker shaft was removed, and cultures contaminated with epithelial cells were discarded.

Before culturing the explants, plastic coverslips or dishes were coated with solutions containing 10 µg/ml murine fibronectin alone or in combination with 5 µg/ml murine tenascin-C, 5 µg/ml murine tenascin-W or a mixture of 5 µg/ml tenascin-C and 5 µg/ml tenascin-W as described previously (Brellier et al., 2012b). Immunocytochemistry, measurements of cell area, and counts of vinculin-positive focal adhesions were performed on stem cells following methods described previously (Brellier et al., 2012b).

Ki-67 proliferation assay

To measure cell proliferation of primary cultures, whisker follicle stem cells were processed for immunocytochemistry (see above). Primary antibodies were rabbit monoclonal anti-Ki-67 (Fisher Scientific, Pittsburgh, PA; clone SP6) and rat anti-mouse CD34, and the secondary antibodies were Alexa Fluor 568 goat anti-rabbit and Alexa Fluor 488 goat anti-rat Ig. Four fields were imaged from each culture and the total number of anti-Ki-67-positive cells was divided by the number of DAPI-positive nuclei to determine the Ki-67 proliferation index (Ostertag et al., 1987). All experiments were conducted three to five times, and the average proliferation index was calculated. Statistical differences were determined with a Student's t-test (equal variance, two-sided).

Acknowledgments

The authors would like to thank Brad Shibata, Tom Blankenship and Paul FitzGerald for assistance and advice.

Footnotes

Author contributions

R.P.T., J.C.S. and R.C.-E. conceived the experiments and prepared the manuscript. R.P.T and J.F. carried out the whisker follicle explant experiments, and R.P.T. carried out the tissue sectioning, immunohistochemistry and immunocytochemistry. The work was conducted jointly in the laboratories of R.P.T. and R.C.-E.

Funding

R.C.-E., J.F. and J.C.S. acknowledge support from the Swiss National Science Foundation [grant numbers 31003A_135584 and 310030_125397].

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